The nervous system is derived from multipotential precursor cells that maintain a closely regulated inverse relationship between cell proliferation and differentiation, as demonstrated in FIG. 1, which employs tissue culture of a neural tumor cell line. In the central nervous system, these precursor cells commit to a specific differentiation pathway shortly after their last mitosis. In the peripheral nervous system, sensory neurons differentiate following withdrawal from the cell cycle, but sympathetic neuroblasts begin to differentiate while still mitotically active. The molecular basis of the coupling between neuronal differentiation and cell proliferation is a problem of current interest which has been extensively studied using cell lines derived from neural tumors. The coupling between neuronal differentiation and cell proliferation is highly relevant to the etiology of neural tumors, in which the regulation of these two cell processes is disrupted.
Neuronal differentiation is induced and maintained by proteinaceous growth factors known as the neurotropins. Several neurotropins are known at this time: nerve growth factor (NGF), brain derived growth factor (BDNF) and neurotrophin-3 (NT-3). Neurotrophins mediate differentiation by binding to and activating high-affinity (K.sub.d.apprxeq.10.sup.-11 M) receptor tyrosine kinases called Trk receptors which appear to transduce most of their biological actions (Chao (1992) Neuron 9:583-93; Hosang et al. (1985) J. Biol. Chem. 260:655-62; Schecter et al. (1981) Cell 24:867-74; and Sutter et al. (1979) J. Biol. Chem. 254:5972-82; Kaplan et al. (1991) Science 252:554-558; Klein et al. (1991) Cell 65:189-197). Activated TrkA is critical for initiating NGF signal transduction, while BDNF binds to a closely related TrkB receptor and NT-3 binds to the TrkC receptor.
Transmembrane receptor tyrosine kinases (RTKs) such as the Trk receptors generally function as molecular switches for transduction of signals from a cell's extracellular milieu, across the cell membrane, into the cytoplasm, and ultimately into the nucleus. Extracellular binding of a cognate ligand to its RTK results in receptor dimerization and autophosphorylation, followed by tyrosine phosphorylation of a specific subset of cellular protein substrates. Ultimately DNA synthesis and cell proliferation or differentiation results from signal transduction via an RTK. A large number of RTKs are known, for example, the epidermal growth factor receptor (EGFR), the platelet derived growth factor receptor (PDGFR), the macrophage colony stimulating factor receptor (CSF-1R), the various fibroblast growth factor receptors (FGFR), the insulin receptor, and the like. The EGFR and PDGFR are known to be involved in glioma growth and progression (see, e.g., Agosti et al. (1992) Virchows Archiv. A. Pathol. Anat. 420:321-5; Torp et al. (1991) Cancer Immunol. Immunother. 33:61-4; Fleming et al. (1992) Cancer Res. 52:4550-3. Additionally both EGF and PDGF tend to promote motility in in vitro assays (compared to NGF which tends to inhibit motility) (Chicoine et al. (1995) Neurosurg. 36:1165-71). Chimeric receptors of EGF and PDGF extracellular domains with the TrkA intracellular domain have been reported (Obermeier et al. (1993) EMBO J 12:933-41) and shown to autophosphorylate and initiate signal transduction in response to EGF and PDGF, respectively.
Recent clinical studies suggest that the TrkA receptor plays a critical role in neuroblastoma, one of the most common pediatric solid tumors. Patients whose tumors express significant levels of TrkA have a good chance for survival, while patients whose tumors lack TrkA respond poorly to therapy (Kogner et al. (1993) Cancer Res. 53:2044-2050; Nakagawara et al. (1993) New Engl. J. Med. 328:847-854; Suzuki et al. (1993) J. Natl. Cancer Inst. 85:377-384). Neuroblastoma frequently occurs during infancy, with the primary lesion in the adrenals and sympathetic chain and metastases to lymph nodes, liver, skin, and bone marrow. This tumor is difficult to treat as common modes of chemotherapy have harsh side effects on normal infant tissue. A variety of modalities have been used to treat neuroblastoma, such as surgery, radiotherapy, and chemotherapy, with varying degrees of success. For many patients, neuroblastoma continues to be fatal.
Cells within neuroblastoma tumors resemble those found in normally developing tissue of the sympathetic nervous system. Neuroblastomas may contain undifferentiated, closely packed spheroidal cells that closely resemble migrating neural crest cells of early embryos (neuroblasts), along with more differentiated cells whose immature nerve fibers tangle, thereby forming a rosette which is the first recognizable sign of neuronal differentiation. Some neuroblastomas undergo spontaneous regression or maturation to benign ganglioneuromas. The similarity of neuroblastoma cells to neuroblasts and the ability of neuroblastoma cells to spontaneously mature to a more benign form indicate that the disease may originate as the result of a block of differentiation of a sympathetic precursor cell.
Another neural tumor, glioma, is a family of cancers comprising the most common adult-onset neurological neoplasms such as malignant astrocytoma (or glioblastoma or malignant glioma), oligodendroglioma, and ependymoma, along with the juvenile onset neoplasms such as juvenile pilocystic astrocytoma (JPA) and the uncommon gangliogliomas. Expression of functional Trk receptors has not been reported for gliomas (Oelmann et al. (1995) Cancer Res. 55:2212-9), though neurotrophin production occurs with fairly high frequency. NGF is secreted by many glioma cell lines (Arnason et al. (1974) J. Clin. Invest. 53:2a; Longo et al. (1974) Proc. Natl. Acad. Sci. USA 71:2347-9; and Reynolds et al. (1981) J. Neurosci. Res. 6:319-25).
While most gliomas are difficult to treat and are ultimately untreatable, there are a few uncommon forms of glioma which are neither aggressive nor invasive. In fact, some patients with these rare gliomas can be followed without treatment for years; others can be effectively treated and even cured with surgery alone. Two types of glioma that behave in such a benign fashion are the uncommon gangliogliomas and JPA. The former tumor, composed of both neoplastic astrocytes and neurons, tends to occur in the temporal lobe of children and behaves very indolently. JPA, characteristically identified histologically by the presence of Rosenthal fibers and microcystic changes, can present as a large cystic mass that often produces symptoms by compressing neighboring structures and causing hydrocephalus. Nevertheless, they are curable by surgery alone, even when they attain significant size. JPA appears to be incapable of invading surrounding tissues.
In contrast, malignant glioma cells produce very invasive brain tumors with infiltration of both white and grey matter (Bjerkvig et al. (1986) Cancer Res. 46:4071-912). At the time of diagnosis, microscopic extension through much of the neural axis by malignant glioma is the rule (Burger et al. (1980) Cancer 46:1179-86; Kelly et al. (1987) J. Neurosurg. 66:865-74; Moser (1988) Cancer 62:381-90; and Salazar et al. (1976) Int. J. Radiat. Oncol. Biol. Phys. 1:627-37). Extension by motile invading cells underlies the incurability by surgery of most gliomas, even when they appear small and restricted in nature. Because gliomas are believed to arise from transformed astrocytes or their immediate precursors, glioma differentiation therapy has been primarily directed at increasing astrocytic differentiation, despite the observation that increasing glial fibrillary acidic protein (GFAP), a specific astrocytic marker, does not correlate with prognosis of these tumors (Delpech et al. (1978) Br. J. Cancer 37:33-40; Eng et al. (1978) J. Histochem. Cytochem. 26:513-23; and Chambers et al. (1991) J. Veuro-Oncol. 11:43-8). In fact, no significant therapeutic advances have been made in treatment of malignant gliomas since the landmark BTCG studies over 15 years ago demonstrated a survival advantage for patients with malignant gliomas who received radiation and single agent chemotherapy (Walker et al. (1978) J. Neurosurg. 49:333-43; and Walker et al. (1980) NEJM 303:1323-9). Cutting edge molecular technologies have led to a better understanding of glioma biology but have not as yet yielded clinical dividends. The median survival for patients with glioblastoma multiforme remains in the range of one year.
Glioma cells for the most part resemble normal glia and are frequently used by neurobiologists as paradigms for glial cells. Glioma tumor progression can be envisioned as an adaptation to local environmental changes or regulatory imbalances. In order for glioma cells to invade normal brain, they must escape from the parent tumor into the surrounding extracellular matrix, hydrolyze matrix components, and migrate through the matrix (Chicoine et al. (1 995) J. Neurosurg. 83:665-71). Glioma invasion into normal brain tissue thus represents the culmination of a series of events involving cell-cell interactions, tumor cell proteinases, adhesion molecules and chemoattractants.
A large number of cell lines have been developed from neuroblastomas (see, e.g., Chen, et al. (1990) Cell Growth and Differentiation 1: 79-85). For example, the SH-SY5Y line was developed from a bone marrow biopsy of a neuroblastoma patient whose primary thoracic tumor had metastasized (Biedler, et al. (1978) Cancer Res. 38, 3751-3757). The LAN5 cell line was similarly developed from a primary tumor (Seeger, et al. (1982), J. Immunol. 128: 983-989). The HTLA230 cell line, isolated from a patient with Stage IV neuroblastoma, has been employed to demonstrate the response of TrkA-expressing neuroblastoma cells to nerve growth factor, both in vitro and in vivo (Matsushima et al. (1993) Mol. Cell. Biol. 13, 7447-7456).
A number of different glioma cell lines have been used to study tumorigenicity, for example, human U-87 and U-373, and rat RT-9 and C6. The C6 rat glioma cell line is well characterized (McKeever et al. (1987) Am. J. Pathol. 127:358-72; and Peterson et al. (1994) J. Neurosurg. 80:865-75), grows well in vivo in non-immunosuppressed rats, and has been recognized as an experimental model of human glioblastoma multiforme (GBM) (Kaye et al. (1986) Cancer Res. 46:1367-73). After implantation, C6 cells also rapidly and extensively invade rat central nervous system (Chicoine et al. supra. Bernstein et al. (1990) Neurosurg. 26:622-8; and Bernstein et al. (1991) Neurosurg. 28:652-8), and movement of homografted C6 cells in brain suggests these cells actively migrate as individual cells in addition to invading as a mass (Bernstein et al. (1991) Neurosurg. 28:652-8). The capacity of C6 cells to aggressively invade both en masse and as single cells is very reminiscent of the clinical situation. Recently, C6 cells which do not express Trk receptors nor respond to NGF were transfected with a trkA-encoding vector (Colangelo et al. (1994) Glia 12:117-27). Exposure of transfected cells to NGF resulted in increased induction of tyrosine phosphorylation of gp140.sup.trk, induction of c-fos mRNA and morphologic changes and (weakly) induction of cell growth.
De Ridder (Neurosurg. (1992) 31:1043-8) demonstrated that in vitro invasiveness of cells derived from primary brain tumor explants correlates with clinical malignant behavior. Additionally, a variety of agents have been shown to stimulate in vitro motile responses including host-derived scatter factors (Ohnishi et al. (1993) Biochem. Biophys. Res. Commun. 193:518-25), growth factors, components of the extracellular matrix and tumor secreted factors (Lund-Johansen et al. (1990) Cancer Res. 50:6039-44; and Chicoine et al. (1995) Neurosurg. 36:1165-71).
The side effects of known brain cancer therapy methods necessitate development of "natural" but highly-specific pharmaceutical treatments, such as various substances that promote differentiation of proliferating neuroblastic cells. At this time, however, none of the candidates for such natural therapeutic approaches have proven successful. Accordingly, methods are needed for inducing malignant brain tumors to become less motile, less proliferative, less invasive, and hence less malignant, or for inducing cells within such tumors to become less tumorigenic, for example, by differentiating into their non-malignant counterparts.